The Molecular Core of the YCCMD provides support for a number of specialized methodologies and technical assistance in essential molecular techniques. The creation of animal models of gene dysregulation and the characterization of target gene expression is fully supported the Molecular Core, with further analysis both in vivo and in vitro provided by the Cell and Physiology Cores. Aim 1. To provide as services, a) the construction of transgenes and gene disruption vectors, and b) the identification of genetically altered or naturally occurring mutant mice. This includes constitutive transgenes, inducible transgenes using the tetracycline transactivator system, conventional gene ablation vectors, conditional gene targeting vectors (using Cre-loxP or Flp-frf approaches), gene knock-in with expression tags (IRES-LacZ or -GFP) and inducible gene targeting (with Cre-ER or Cre-PR fusions). Genotyping is carried out by PCR and/or Southern blot analysis of tail DMA and/or ES cell DMA. Aim 2. To provide as services, a) the localization of gene expression by in situ hybridization, and b) the quantitation of gene expression by quantitative, real-time RT-PCR. For expression localization, this includes the dissection and fixation of tissues, embedding in paraffin or OCT, sectioning by microtome or cryostat, preparation of riboprobes or oligoprobes, hybridization, emulsion autoradiography and image analysis. For quantitation of gene expression, this includes tissue dissection, RNA preparation, RT primer selection and PCR primer design. Aim 3. To provide technical assistance, training and resources for essential molecular methods, including: RNA and genomic DNA preparation and analysis; stable or transient cell transfection; reporter gene analysis; PCR-related techniques, such as mutagenesis, cloning, overlap extension and inverse PCR; siRNA design, Dicer pools and shRNA (short hairpin) vectors and viruses for projects that require the suppression of gene expression by RNA interference; and BAG recombineering.